Review

What is new in Diagnosis of Tuberculosis

¹Shashi Prakash, ¹Shivanshu Singh, ²Divya Khanna, ³Anuradha Khanna, ¹A. K. Khanna

• ¹Department of Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
• ²Department of Community Medicine, King Georges Medical University, Lucknow, India
• ³Department of Obstetrics & Gynecology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

• Submitted: November 3, 2012
• Accepted: December 6, 2012
• Published: December 8, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tuberculosis is an infectious disease caused by acid-fast bacillus Mycobacterium tuberculosis. It most commonly affects the pulmonary and less commonly the extra-pulmonary sites. It is one of the leading causes of global mortality and morbidity. Despite being such a common disease, the accurate, reliable and rapid diagnosis of tuberculosis has always been challenging even after years of research. Recently, the diagnosis of tuberculosis has reached a new horizon with the advent of molecular diagnostic methods. Still today, mycobacterial culture remains the reference standard test and majority of the newer approaches serve as a compliment to sputum smear microscopy and culture. Rapid diagnosis of drug resistance has opened new channels in the management of tuberculosis demanding the need for the development of effective treatment against MDR and XDR tuberculosis.

Introduction

Tuberculosis is one of the leading causes of mortality and morbidity globally. In 2010, there was an estimated 8.5–9.2 million cases and 1.2–1.5 million deaths (including deaths from TB among HIV-positive people) worldwide [1]. Most of the estimated number of cases in 2010 occurred in Asia (59%) and Africa (26%); smaller proportions of cases occurred in the Eastern Mediterranean Region (7%), the European Region (5%) and the Region of the Americas (3%). The five countries with the largest number of incident cases in 2010 were India (2.0 million–2.5 million), China (0.9 million–1.2 million), South Africa (0.40 million–0.59 million), Indonesia (0.37 million–0.54 million) and Pakistan (0.33 million–0.48 million). India alone accounted for an estimated one quarter (26%) of all TB cases worldwide, and China and India combined accounted for 38%.

This global burden demands the development of diagnostic tests that provide early, rapid, sensitive, specific and cost effective approach for effective management of cases. Conventional methods include the sputum smear examination (sensitivity of only 44% for all new cases [2], tuberculin skin testing, chest X ray, and mycobacterial culture. Though culture is the most definitive test, it takes around two or more weeks [3], thereby delaying the diagnosis. To control the global burden of this widespread disease, newer diagnostic methods are being innovated and tested for their utility. Advances have been made in the molecular diagnosis based on nucleic acid amplification for earlier and specific diagnosis of Mycobacterium tuberculosis directly on the clinical specimen [4]. A good combination of diagnosis and treatment modalities is essential for effective control of tuberculosis. This review article will discuss the recent trends in the diagnosis of latent and active tuberculosis infection considering the pros and cons of newer diagnostic methods.

Diagnosis of Latent TB infection

Although tuberculin skin testing (TST) has been in use for several years, it has major limitations: sensitivity is very low in infants, toddlers [5] and old age [6], false positivity due to antigenic cross reaction with nontuberculous mycobacterial [7] or previous BCG vaccination [8], false negative results in cases of acquired immunodeficiency (e.g. HIV infection) [9], corticosteroid treatment [10], chronic renal failure [11], malnourishment [12,13], cancer [14] or severe illness (including active form of tuberculosis) [15], booster effect of previous TST, errors in administration of injection, painful skin injections, subjective variation in result interpretation and the need for repeated visits to the clinic. With the advancements in the research of tubercular genomic architecture, in vitro tests have been developed. Interferon gamma release assays (IGRA) measure the release of IFN gamma by the sensitized T cells when exposed to tubercular antigens. Although, earlier PPD (purified protein derivative) was used as the antigen but currently it has been replaced by recombinant early secretory antigenic target (ESAT) – 6 and culture filtrate protein (CFP) – 10 [16]. These antigens are encoded by the genes in the region of difference (RD1) segment of M. tuberculosis genome. These antigens do not have cross reactivity with nontuberculous mycobacterial infection. IGRAs are more sensitive and specific than TST (80-95% vs. 75-90% and 95-100% vs. 70-95% respectively) [17].

Commercially available IFN- γ based test kits include: QuantiFERON- TB Gold(Cellestis Ltd., Carnegie, Australia) kit [18] and the T SPOT – TB (Oxford Immunotec, Oxford, UK) [19]. The US-FDA has approved the 24-well culture plate format of QuantiFERON – TB Gold [20]. It’s another simplified in-tube format is not FDA approved. The T SPOT- TB is CE marked for use in Europe. QuantiFERON- TB Gold is an ELISA based test and uses whole blood while the T SPOT – TB test counts the cells releasing IFN- γ visualized as spots in the ELISPOT technique (enzyme-linked Immunospot technique).

Since the specificity of the IGRA is very high, it shall be helpful in LTBI detection in resource rich areas having low prevalence of tuberculosis where cross reactivity due to BCG vaccination and non tubercular mycobacterial infection may result in false positive TST [21]. Also, because of the lack of booster effect in IGRA, they can be used for serial testing of health care workers [21].

Despite the presence of such potential advantages, many issues regarding IGRAs are still under study. Whether the association between IFN – γ response to ESAT-6 has any predictive value for future progression to active tuberculosis still needs to be largely determined, though limited evidence is present [22]. Research is going on to determine the impact of anti tubercular treatment and IFN γ responses to ESAT-6, thus searching for its role in monitoring the treatment outcome. Several studies have shown discordance between results of IFN – γ and TST [23]. Also, more study needs to be done on the role and utility of this test in the subgroup of immune compromised patients (e.g. HIV infection), cases of extra pulmonary tuberculosis, children and population in high incidence countries [23]. In resource poor, high incidence areas, the material cost and the need for the laboratory infrastructure is another problem. Thus, studies are required to assess the cost effectiveness of this test in clinical and public health settings [24].

Diagnosis of active tuberculosis

The diagnosis of tuberculosis relies on clinical suspicion, imaging studies (though not pathognomonic) or microbiological diagnosis. Imaging modality commonly used is the chest X-ray, although CT scan and more recently, PET scans have been used for disease monitoring [25]. Microbiological diagnosis includes the conventional sputum smear examination, bacterial culture and molecular methods like Nucleic Acid Amplification Tests (NAAT). Apart from these, many newer methods are under investigations like FACS (Fluorescence- activated cell sorting), mycobacterial proteomics, gold nano particle probe assay, breath test and many more down the lane.

Clinical features

The clinical features of both pulmonary as well as extrapulmonary tuberculosis are often nonspecific. In cases of childhood tuberculosis, the usual presenting symptoms are fever, cough, weight loss, night sweats, fatigue, tuberculosis contact, malnutrition, lymphadenopathy, organomegaly [26]. Because of the paucibacillary nature, confirmed bacteriological diagnosis is possible in only 30-40% of affected children [27,28]. Tuberculosis in the elderly usually presents in an atypical manner [29]. Dyspnoea is more frequent while fever, sweating and haemoptysis are less common [30]. Presence of concomitant HIV infection further complicates the issue. Incidence of extrapulmonary and miliary tuberculosis is commoner in the old age [31]. In 2007, out the 1.76 million cases who expired due to tuberculosis, 4,55,000 were seropositive [2]. The ‘delay in diagnosis’ [3] of tuberculosis is often because of the lack of suspicion, especially in the low prevalence population. The ‘delay in the diagnosis’ is one of the basic indicators of quality of tuberculosis diagnosis. Delay may occur at two levels: i} patient delay: It is the time from the onset of symptoms to the first visit in the hospital, ii} health system’s delay: the time from the first visit of the patient until the establishment of the diagnosis of tuberculosis. Several studies have shown that the cause of delay includes insufficient medical workup like sputum examination for AFB [32], too much dependence on X-rays while giving less attention to bacteriological diagnosis [33].

Recent trends in the imaging diagnosis of tuberculosis

Still today, chest X-ray is the most commonly performed imaging investigation in a tuberculosis suspect. Earlier, the x-ray appearance of were classified based the stage of disease viz. primary or secondary tuberculosis while the recent evidence suggests that the X-ray features depend upon the immune status of the patient. Cases who are immune competent have the appearance of secondary tuberculosis which include focal heterogeneous consolidation in the apical and posterior segment of the superior lobe and the superior segment of the lower lobe, poorly defined nodules, linear opacities and cavitations. In cases of immunocompromised individuals, findings similar to that of primary tuberculosis are seen which include unilateral hilar lymphadenopathy, parenchymal consolidation and/or pleural effusion [25, 33-37]. On the computed tomography, features suggestive of pulmonary tuberculosis include cavitation, centrilobular nodule and the “tree-in-bud” appearance [38]. High resolution CT scans are more sensitive than the conventional chest X-rays for the detection of early parenchymal lesions, mediastinal lymph node status and disease activity of tuberculosis [39,40]. PET scans have also found a role in the diagnosis of tuberculosis. Serial pulmonary [(18F)]- 2-fluoro-deoxy-D-glucose positron emission tomography is emerging as a potential tool for monitoring the disease activity and response to anti tubercular treatment [41,42]. Cost- effectiveness of this technique is still an important issue.

Recent trends in the microbiological diagnosis

Microbiological diagnosis is more specific than the imaging studies and the clinical symptoms. Different methods include the microscopic demonstration of the acid-fast bacilli in the clinical specimen (e.g. sputum), the mycobacterial culture and molecular techniques including the nucleic acid amplification tests. There have been certain advancements in these diagnostic methods in terms of increased sensitivity, better specificity and rapidity of diagnosis.

Advances in the microscopic examination

Being relatively easy, cheap and quick to perform [43], smear examination under light microscopy after acid-fast staining is still the most common bacteriological diagnostic method used in resource poor regions. However, its poor sensitivity (45-80%) has limited its usefulness in areas having lower incidence, extrapulmonary forms and HIV-infected cases [43]. Although relatively specific, the positive predictive value of the test is lower (50-80%) in areas having high incidence of nontuberculous mycobacteria in clinical isolates [44,45,46]. As compared to conventional microscopy, fluorescent microscopy has a better sensitivity [47]. Concern about the loss of specificity, especially in the developing world is still there. Use of ultra-bright light emitting diode (Lumintm, LifeEnergy (R), Germany) further improves the bacillary detection [48]. Studies regarding the processing of the specimen have shown that centrifugation combined with a chemical method improves the sensitivity [49]. A sample for smear is usually taken at three times. However, in laboratories with standard quality control, the utility of third sample has been challenged [50-53]. Patients who are not able to produce sputum may be subjected to sputum induction by any method to increase the sensitivity of the smear examination [3]. Commonly used method is inhalation of 3% NaCl solution. ‘Lung flute’ is a small self-powered audio device that generates sound waves, which vibrate in tracheobronchial secretions, thereby allowing the sputum sampling [54]. In developed and resource-rich regions, fibre-optic bronchoscopy with broncho-alveolar lavage or gastric washings are sometimes used in patients who are not able to produce sputum. Other clinical specimens for extrapulmonary tuberculosis include the formalin preserved biopsy material for histopathological analysis and saline preserved for microbiological tests, unprocessed fluid from pleural effusion, ascites, cerebrospinal fluid, urine, and stool [3]. Heparinised or lithium citrate tubes are used for blood and bone marrow specimen [3]. The microscopic examination of the smear has become so important that quality assurance of each laboratory has been advocated [55]. Many researchers have focused on new tools and methods to improve the sensitivity and/or efficiency of microscopy, three of which are: Front-loaded smear microscopy, LED fluorescence microscopy and bleach microscopy.

Front-loaded smear microscopy

Microscopy services currently require patients to make repeat visits to the healthcare facility. This is associated with considerable patient costs and patient drop-out during diagnosis. The new WHO definition of a smear positive case does not require confirmatory smears. This allows patients to be diagnosed on a single smear. Two smears have been shown to diagnose 95-98% of patients. In front-loaded microscopy the first two specimens are collected and examined on the same day a patient presents. Patients with negative smears can be asked to return with a morning specimen the next day, depending on whether routine services are based on two or three specimens being examined.

Potential advantages: The costs for visits incurred by patients, particularly by poor patients, are often prohibitive. This new method intends to reduce these costs and the considerable proportion of patients dropping out of the diagnostic process. TB patients can be offered treatment on the day they present.

Considerations: In addition to this method, programmes can seek ways to provide better follow-up in order to reduce patient drop-out. Data on the impact and effectiveness of this method is limited; studies to validate the method are currently in progress. To make best use of this method, clinics may have to reorganize their clinical and laboratory services so that people have both their smears examined on the same day – making it possible to put them onto treatment rapidly.

Requirements: Does not require any equipment or biosafety measures beyond that already required for direct smear microscopy. Some supervised training of existing smear microscopy laboratory technicians is required to change from a two-day to one-day sampling. Laboratories performing front-loaded microscopy require the equipment and consumables needed for traditional smear microscopy. National guidelines for TB control, forms and registers may need revision to account for changed protocols.

LED fluorescence microscopy

The use of fluorescence microscopy (FM) could potentially increase the sensitivity of smear microscopy and improve the efficiency of facilities. These microscopes allow a much larger area of the smear to be seen, resulting in more rapid examination of the specimen (up to four times faster) and making it easier to count bacilli.

Potential advantages: Light-emitting diodes (LEDs) are more sustainable and user-friendly than the quartz-halogen lamps or high-pressure mercury vapour lamps typically used in FM: powered correctly, they have an extremely long life expectancy (10,000 hours versus 200 for a conventional mercury lamp). Unlike the light emitted by mercury vapour lamps, LEDs do not produce ultra-violet (UV) lights (a cause of concern to many users). LEDs significantly decrease the instrument’s power consumption, allowing long-lasting battery operation. LED technology allows FM at a much lower cost than conventional FM.

Considerations: In some countries, it may be difficult to find the LED lamps required. Also, the Royal Blue colour LED lamp which works best for FM was only very recently developed and may not be widely available. Some issues with the stability of reagents under field conditions, and the stability of stained smears for blinded rechecking, have been reported. These need further investigation. Proper training is critical; it must be carefully considered and carried out properly. The sensitivity, specificity, cost-effectiveness and cost-benefit of this approach have not yet been adequately established. International guidance on quality assurance for FM does not currently exist.

Requirements: Apart from an LED microscope (or an adaptor to convert an existing microscope to LED FM) and stains for FM, this technology does not require equipment or biosafety measures beyond that already required for smear microscopy. Training of existing smear microscopy laboratory technicians is required to perform FM. LED FM may be powered by electricity or batteries. There are various manufacturers of LED fluorescence microscopes or adaptors converting standard light microscopes to LED fluorescence microscopes. These are proprietary products and countries may find it difficult to find local/national suppliers, so establishing a long-term support agreement with an international supplier is critical. Some LED equipment (the Fraen and the Lumin) can be used as add-ons to existing microscopes, while others (the iLED) are complete microscope kits.

Sodium hypochlorite (bleach) microscopy

The digestion of sputum with household bleach prior to sputum smear preparation and microscopy has been reported to be an effective, simple method to improve the yield of smear microscopy even in high HIV prevalence settings. Progress on the development of a bleach microscopy method has been complicated by the wide heterogeneity, and lack of standardization, in methods described. A standardized bleach method, the Mathare Sodium Hypochlorite (MaSH) method, has recently been developed and evaluated in MSF-sponsored studies in Mathare, Nairobi. The addition of a standardized sodium hypochlorite solution to sputum followed by overnight sedimentation resulted in a 15% increase in TB cases detected. The MaSH Method is now being evaluated by TDR under operational conditions in large multi-country studies.

Recent trends in the mycobacterial culture techniques

The mycobacterial culture is still considered to be the gold standard method due to its excellent sensitivity. Also, further studies can be done on the isolated mycobacteria (identification, sensitivity and epidemiological typing) [46,47]. The major limitation is the slow growth of mycobacterium, thereby causing a delay in the diagnosis. Advancements have been made in the culture with the use of new liquid media and automated systems such as Bactec 460TB (Becton Dickinson Diagnostics, Sparks, USA), MB/BacT ALERT (bioMérieux, Marcyl’Etoile, France), MGIT 960 (Becton Dickinson Diagnostics) and Versa TREK (Trek Diagnostic System, Westlake, USA) [43]. The average time required for detection is 12.9 days by BACTEC MGIT960 and 15 days with BACTEC 460 as compared to 27 days with Lowenstein Jensen solid medium [56]. Thus, WHO has now recommended the use of liquid tuberculosis culture and drug susceptibility testing for M. tuberculosis in low-resource settings [57]. Liquid culture systems reduce the delays in obtaining positive cultures. The rapid growth in liquid culture results in reduced delays for DSTs also, compared with conventional solid media. Liquid systems are more sensitive for detection of mycobacteria and may increase the case yield by 10% over solid media. With increased sensitivity and reduced delays, liquid systems may contribute significantly to improved patient management. It should be noted, however, that just because a tool relies on a liquid culture medium, does not necessarily mean it is endorsed by WHO. The Expert Meeting on liquid culture convened by WHO only reviewed evidence on the use of commercial broth-based culture systems, and recommendations only pertain to these systems. Four new tools for improving culture diagnosis and DST are: Liquid culture: Manual liquid culture technique and commercial broth-based culture systems, Solid culture: Nitrate reduction assay and Thin layer agar culture, and Colorimetric redox indicators.

Liquid culture: Commercial broth-based culture systems

New tools can detect fluorescence in a liquid culture medium, enriched with oxygen, to indicate the presence of bacteria. As bacteria grow in the culture, the oxygen is utilized, causing it to be fluorescent when placed under UV light. Methods for testing for drug susceptibility follow the same principle but use two culture samples: one with a drug added and one without the drug (a growth control). If the test drug is active against the TB bacteria, it will inhibit growth and suppress fluorescence. In the manual system, a technician visually identifies fluorescence using a hand-held UV lamp. Automated systems have the capacity for up to 960 cultures at a time.

Potential advantages: Such systems have been thoroughly evaluated in clinical settings and approved for use in industrialised countries for years. Liquid culture systems provide results significantly faster than solid culture techniques: on average diagnosis can be performed in 7-14 days (up to 42 days for a negative result). DST takes on average 7-14 days (range 4-14 days) after the initial culture. Automated systems can benefit laboratories with a high workload and provide standardized reading of samples. Such systems have a sensitivity and specificity of nearly 100% (slightly less for manual systems). Studies have shown that both automated and manual systems perform well in detection of isoniazid and rifampicin susceptibility, but are not as effective for ethambutol and streptomycin.

Considerations: The machine (required for the automated system) and the growth indicator tubes (required for both manual and automated systems) are costly, although a “Developing country” price has been negotiated for MGIT by FIND. Manual systems typically require additional manipulation and use of a hand-held UV lamp. Liquid media are more prone to contamination than solid media, leading to invalid tests unless carefully controlled. Automated machines must be maintained, requiring on-site technical support from manufacturers or their agents.

Requirements: As with all TB culture systems, BSL-2 facilities are required for processing specimens and inoculating cultures. BSL-3 is required if tubes need to be opened and the cultured organisms manipulated (for speciation or further testing). Since speciation of organisms grown in commercial broth-based culture systems requires tubes to be opened and cultures to be manipulated, BSL-3 facilities are required. Laboratory technicians require training in BSL-3 safety precautions and training in the use of these methods and tools. A stable source of electricity is required for automated systems. Laboratories performing rapid culture in liquid medium require sophisticated technology (such as the BACTEC™ MGIT™ 960 System for the automated Mycobacterium Growth Indicator Tube test) or a hand-held UV lamp (for manual systems) as well as several consumable products for which a supply chain must be established, such as BBL MGIT Indicator Tubes, centrifuge tubes, sodium hydroxide, sodium citrate solution, N-acetyl-L-cysteine powder, phosphate buffer, vortex mixer, incubator, pipettes and pipette tips, sodium sulfite solution, mycobacterial agar or egg-based medium, tissue homogenizer or sterile swab, Normal saline, ATCC reference strains, microscope and materials for staining slides, and blood agar plates. Some methods of rapid culture in liquid medium require proprietary products and countries may find it difficult to find local/national suppliers, so establishing long-term support agreements with international suppliers is critical.

Colorimetric redox indicators

Colorimetric methods are based on the reduction of an indicator solution added to a liquid culture medium after TB organisms have been exposed to different antibiotics. Isoniazid and rifampicin resistance is detected by a change in colour of the indicator.

Potential advantages: There is limited evidence that colorimetric methods are highly sensitive (about 95%) and specific for the detection of MDR-TB and faster than conventional solid or liquid culture DST methods (first results between 7 and 14 days after culturing). Colorimetric methods do not require sophisticated equipment and are therefore less expensive to perform than some other liquid culture techniques for DST.

Considerations: Colorimetric methods cost approximately the same as solid culture techniques for DST. Additional studies and cost-effectiveness analyses are required to validate this technique and determine if appropriate for large-scale implementation in countries with a high prevalence of MDR-TB.

Requirements: As with all TB culture systems, BSL-2 facilities are required for processing specimens and inoculating cultures. BLS-3 facilities are required if tubes need to be opened and the cultured organisms manipulated (for speciation or further testing). Since the colorimetric test requires that the microtitre plates be opened and indicator solution pipetted into the wells, BLS-3 facilities are required. Laboratory technicians require training in BSL-3 safety procedures and in the colorimetric DST methodology. Laboratories performing colorimetric DST require the equipment (centrifuge, balance, Bunsen burner, freezer, incubator, refrigerator, vortex mixer and water distillator) and consumables (antiseptics and dispensing syringes) required to perform liquid culture (see above). Furthermore, the test requires equipment (microtitre plates) and consumable products for which a supply chain must be established, including Middle brook 7H9 broth and a redox indicator (MTT solution or resazurin).

New solid culture methods: Nitrate reduction assay (NRA), e.g. Griess method

Solid culture technique measures nitrate reduction to indicate resistance to isoniazid and rifampicin. This technique is based on the property of TB to reduce nitrate to nitrite, which is revealed as a colour change of the culture media.

Potential advantages: The nitrate reductase assay (NRA) is less expensive to perform than liquid culture techniques for DST. Furthermore, since the NRA method makes use of the recognition of nitrate reduction as a sign of growth, results are acquired earlier than by eye examination of colonies in solid culture. Studies have shown that the specificity and sensitivity of NRA were comparable to traditional solid culture methods for DST of isoniazid and rifampicin. It uses solid media, which may be safer than liquid media. NRA does not need sophisticated equipment, is not complex to perform and could therefore be appropriate for laboratories with limited resources.

Considerations: The culture is killed by the mix reagent used to develop the assay, requiring that multiple cultures be prepared if comparative testing will be performed. Only fresh cultures must be used (<14 days). Additional studies and cost-effectiveness analyses are required to validate this technique and determine if it is appropriate for large-scale implementation in countries with a high prevalence of MDR-TB.

Requirements: As with all TB culture systems, BSL-2 facilities are required for processing specimens and inoculating cultures. BSL-3 facilities are required if tubes need to be opened and the cultured organisms manipulated, such as for speciation or further testing. Since speciation of M. tuberculosis growing in NRA cultures is done by visualization of a colour change in the tube (without opening them) BSL-2 facilities may be sufficient. Laboratory technicians require training in appropriate safety precautions and in how to perform NRAs. Laboratories performing NRA require the equipment and consumables required to perform solid culture (see above). Furthermore, the test itself requires several consumable products for which a supply chain must be established, including sodium nitrate and either hydrochloric acid, sulfanilamide and N-naphthylethylene-diamine (if the test will be performed with liquid reagents) or sulfanilic acid, N-(1-naphthyl)-ethylenediamine dihydrochloride and tartaric acid (if the test will be performed with a crystalline reagent).

New solid culture methods: Thin layer agar culture (TLA)

The technique uses a standard microscope to simultaneously detect TB bacteria and indicate isoniazid and rifampicin resistance. Plates with a thin layer of agar medium are incubated and examined microscopically on alternate days for the first two weeks and less frequently thereafter.

Potential advantages: Inexpensive compared to other culture techniques. Test can be done with a standard light microscope. At least one study has demonstrated that this technique provides results faster (in about 11 days) than traditional solid culture using Lowenstein–Jensen medium (26.5 days). Sensitivity obtained with thin layer agar is slightly better than with Lowenstein–Jensen. TB bacteria can be detected in as little as 7 days, with results for DST between 10-15 days. Uses solid media, which may be safer than liquid media. Simpler to manage large numbers of samples than for manual liquid culture.

Considerations: The contamination rate is slightly higher than when using Lowenstein–Jensen medium. Although reportedly faster than traditional solid culture techniques, it is not as fast or as sensitive as liquid culture. Further studies are required to adequately validate this technique.

Requirements: As with all TB culture systems, BSL-2 facilities are required for processing specimens and inoculating cultures. BSL-3 facilities are required if tubes need to be opened and the cultured organisms manipulated, such as for speciation or further testing. Since speciation of M. tuberculosis growing in TLA cultures is done without opening the Petri dishes, BSL-2 facilities may be sufficient. If Petri dishes cannot be adequately sealed, however, then BSL-3 facilities are required. Laboratory technicians require training in the appropriate safety precautions and in the use of thin layer agar cultures. Laboratories performing the thin layer agar technique require the equipment and consumables to perform solid culture (see above), as well as additional consumable products for which a supply chain must be established: OADC, PANTA, Middle brook agar-based medium and Petri dishes.

Role of bacteriophage in cultures

Luciferase Reporter Phage Assay (LRP) and the Phage Amplified Assay (PhaB or MAB) are the two bacteriophage based assays used for rapid detection of tubercular mycobacterial in the culture [58,59,60]. These assays are meant to detect phage-infected mycobacterial cells. In the LRP assay, the genome for the light emitting Luciferase (fflux) enzyme is inserted into the phage genome and the emitted light forms the basis of this test. In PhaB or MAB assay, the presence of viable M. tuberculosis complex cells is looked for after phage amplification (Mycobacteriophage D29) in Mycobacterium smegmatis. LRP has been useful in differentiating M. tuberculosis and NTM from culture and, especially, in susceptibility tests to isoniazid and rifampin. PhaB or MAB has been commercialized (FASTPlaque-TB or the variant PhageTeK MB, Biotec Laboratories Ltd, Ipswich, Suffolk, UK) for diagnosing tuberculosis in respiratory specimens [59], and has also been studied for antimicrobial susceptibility testing in M. tuberculosis [61]. Both techniques are generally quick and simple, requiring little training and technical equipment, and they are relatively inexpensive. However, although they have demonstrated good specificity, several problems of sensitivity have been encountered in most studies [59,60]. The routine application of these techniques is therefore, still pending. The practical utility of these tests is therefore, still to be determined.

The commercially available kits for phage based detection of drug resistance include FASTPlaque-TB (Biotec Laboratories Ltd., Ipswich, UK) assay used directly on sputum specimens. Another variant, the FASTPlaque-TB-MDRi kit, has been made to detect rifampicin resistance in culture isolates [21]. In a meta-analysis on the role of phage based assays for the detection of drug resistance, it has been shown by several studies that the sensitivity and specificity on culture isolates were in the range of 95% or more [60]. However, when tested directly on the clinical specimen, the sensitivity was very low and inconsistent probably due to low bacillary concentration [60]. This is the major drawback since the prerequisite of having a culture isolate delays the diagnosis of drug resistance by the phage based assays.

Molecular methods for the diagnosis of M.tuberculosis - a new horizon

Nucleic Acid Amplification Test

The problem of having a rapid diagnosis received an answer with the application of nucleic acid amplification tests (NAAT). These tests identify the specific nucleic acid sequences of the M.tuberculosis. They are also known as ‘direct amplification test’, since they are performed directly on the clinical specimen e.g. sputum [21]. NAAT tests are classified into commercial kits and in-house assays. Commercial kits are the Amplicor MTB tests (Roche Diagnostic Systems, Branchburg, NJ), the Amplified Mycobacterium tuberculosis Direct Test (MTD) (Gen-Probe, Inc., San Diego, CA), and the BD ProbeTec ET assay (Becton Dickinson Biosciences, Sparks, MD). The Amplicor and MTD tests are FDA approved. In-house tests are laboratory-developed polymerase chain reaction (PCR) assays; they vary greatly in their design and laboratory methods [21]. The results of NAAT can be obtained in a day or two after obtaining the sputum or the bronchoalveolar lavage fluid [3].

The validity of these tests has been extensively reviewed and analysed [63-67]. In cases with a positive sputum smear on the microscopy, the sensitivity of NAAT is more than 95% [68,69]. Therefore, a presumptive diagnosis of tubercular infection can be made early in individuals with a positive AFB sputum smear on microscopy. Also, a negative NAAT on a positive sputum smear indicates that the AFB are non-tubercular mycobacterial species [3]. The sensitivity of these tests is variable in sputum smear negative pulmonary tuberculosis [67]. A negative NAAT test does not rule out tuberculosis in a sputum smear negative case [21]. The specificity of NAAT in cases with negative sputum smear is high in the range of 97-98% [68, 70]. Commercial NAATs can therefore, be confidently used to exclude TB in patients with smear positive samples in whom environmental mycobacterial infection is suspected and to confirm TB in a proportion of smear negative cases [68]. In 2005, Flores and colleagues concluded that the estimates of accuracy of in-house assays were highly inconsistent and the use of IS6110 as an amplification target along with nested PCR methods were associated with higher diagnostic accuracy [63]. Commercial NAA tests have a potential role in confirming tubercular meningitis, however, because of their low sensitivity, they cannot rule out tuberculosis [65]. The in-house PCR based assays have limited clinical utility because of the variability in their test results [21], whereas the commercial assays are more standardized. Regarding extrapulmonary tuberculosis, the NAA tests have low sensitivity and negative predictive value and therefore cannot rule out tuberculosis [21]. The false positive results are found in individuals with past history of tuberculosis and in cases of bronchogenic carcinoma [3]. These tests are not used to monitor the response to anti-tubercular treatment, since they amplify the nucleic acid of both the viable as well as the non-viable bacilli [21].

Role of DNA and Ribosomal rRNA based probes: DNA probes are used to identify tubercular mycobacterial sequences in cultures or directly on the clinical specimen. Commercially marketed probes are available for M. tuberculosis and M. avium [71,72]. The DNA based probes are not very sensitive for detection on direct clinical specimens and require the presence of 10000 organisms for positivity [4]. In contrast, the recently developed ribosomal rRNA based probes are 10-100 times more sensitive than the DNA targeting probes [73].

Role of isothermal amplification technique

Apart from the polymerase chain reaction, another gene amplification technique includes the isothermal amplification technique [4]. It uses enzymes other than the taq polymerase and the entire process of amplification is completed at a single temperature only. Three approaches have shown promising results. The strand displacement amplification (BD ProbeTec ET Direct TB System) [74], isothermal amplification of M. tuberculosis complex rRNA followed by detection of amplicon with acridinium ester-labelled DNA probe [75] and the third method is based on isothermal gene amplification with QB replicase enzyme for production of RNA and has a sensitivity of up to one colony forming unit for M.tuberculosis [76].

Recent trends in line probe assays

Line probe assays are being used for the rapid detection of mutations resulting in the drug resistance. The commonly available kits are Genotype MTBDR assay, Hain Lifescience, Nehren, Germany [77] or INNO-LiPA Rif. TB kit, Innogenetics, Zwijndrecht, Belgium [78]. Line probe assay technology involves the following steps [79]: Initially, DNA extraction from M.tuberculosis isolates or directly from clinical sample. Next, using biotinylated primers, the polymerase chain reaction (PCR) amplification of the resistance-determining region of the gene under suspicion is performed. Following amplification, labelled PCR products are hybridized with specific molecular oligonucleotide probes immobilized on a strip. Captured labelled hybrids are detected by colorimetric development, enabling detection of the presence of M. tuberculosis complex, as well as the presence of wild-type and mutation probes for resistance. If a mutation is present in one of the target regions, the amplicon will not hybridize with the relevant probe. Mutations are therefore detected by lack of binding to wild-type probes, as well as by binding to specific probes for the most commonly occurring mutations. The post-hybridization reaction leads to the development of coloured bands on the strip at the site of probe binding and is observed by eye. Several versions of Genotype MTBDR assay have been developed. The latest one, known as Genotype MTBDRsl assay detects drug resistance to even fluoroquinolones and injectable ATT providing early diagnosis of XDR tuberculosis with a sensitivity of >85% [80]. The Genotype MTBDR plus detects mutations in the rpoBgene, katG gene and the inhA promoter region [3]. Although the sensitivity for rifampicin resistance is similar to conventional DST (>95%), the sensitivity for isoniazid-resistance is low (72-92%) [3]. In 2008, WHO issued a policy statement regarding the role of line probe assays [79] and stated that it cannot completely replace the conventional culture and drug susceptibility testing (DST). DST is still essential to confirm XDR tuberculosis. However, the application of line probe assays for screening MDR-TB may reduce the burden on conventional culture and DST laboratory.

Role of molecular beacons

Molecular beacons are fluorescent labelled, hair-pin-shaped DNA probes. They are added to the real time PCR assay. If the wild type of mycobacterial genomic sequence is there, the beacon unfolds and fluorescence is detected. In case of mutation, the beacon does not unfold and no fluorescence is detected. The sensitivity for rifampicin resistance is 89-98% and specificity 99-100% but sensitivity is very low for isoniazid resistance [81-84].

Recent trends in the immunological diagnosis of tuberculosis

Advances in serology for detection of antigen/antibody of M.tuberculosis

This method depends on the detection of M.tuberculosis specific antibodies (IgG, IgA, IgM), antigens like 38 kDa, 16 kDa, 88 kDa, MPT51, malate synthase, CFP-10, TbF6 polyprotein, antigen 85B, antigen A60, antigen 5, alpha-crystallin, lipoarabinomannan, Rv3425 etc. or mycobacterial protein, lipid or polysaccharides. WHO/TDR evaluated these tests and found that the sensitivity and specificity of the commercially available test were low 1-60% and 53-99% respectively and concluded that none of the assays have performed well enough to replace microscopy [85].

Local immunodiagnosis by IGRA

In cases of active pulmonary tuberculosis, the antigen specific T-cells clonally expand and are concentrated at the site of infection [3]. In such cases, a rapid diagnosis can be made by subjecting the broncho-alveolar lavage to ELISPOT test and it showed a sensitivity of 91% and specificity of 80% respectively in cases of AFB sputum smear negative pulmonary tuberculosis [70]. BAL-ELISPOT is more sensitive than NAA tests for detection of sputum smear negative cases of pulmonary tuberculosis. Ideal scenario for BAL-ELISPOT is an area with low incidence of pulmonary tuberculosis where bronchoscopy is routinely performed and the facility for ELISPOT test is available [3].

Fluorescence activated cell sorting

It is an immunophenotyping technique of the antigen stimulated cells of the broncho-alveolar lavage or sputum cells for rapid diagnosis of tuberculosis in sputum smear negative cases. In this technique, the cells of interest in the mixture are linked with the fluorescent tagged antibody and are subjected to laser beam in a stream and the light causes fluorescence that is picked up by the computer and it decides which cells are to be separated. The problem is that the population of T-cells specific to the region of difference-1 of M.tuberculosis is too low in the sputum to be picked up by the flow cytometry [3]. But, this technique is a promising tool for the further improvement of the local immunodiagnosis of AFB smear negative tuberculosis [86].

Other newer modalities under investigation

Newer biomarkers, e.g. IP-10 and MCP-2 are being investigated. Of the biomarkers studied, MCP-2 and IP-10 have held the promise as the antigen stimulated levels were high in patients and not in controls [87]. IP-10 is an interferon induced protein and is a CXC chemokine while MCP-2 is the monocyte chemoattractant protein and is a CC chemokine. Concepts of proteomics are also being applied for the diagnosis of tuberculosis. Using the support vector machine, it was found that the proteomic profile of a tuberculosis infected subject was different from the control [88]. Diagnostic accuracy was 94% (sensitivity 93.5%, specificity 94.9%) for patients with tuberculosis and was unaffected by HIV status. The sensitivity of urinary lipoarabinomannan tests based on antigen capture ELISA has been found to be somewhat higher in HIV seropositive individuals [89] and thus can be used in combination with sputum-smear test to improve the case detection in such cases.

Table 1- Summary of new technologies for the diagnosis of tuberculosis.

Technology	Description
WHO-endorsed tools (2006-2008)
Liquid culture	Commercial broth-based culture systems detect TB bacteria (manual and automated systems are available); can be configured for DST.
Molecular line probe assay	Strip test simultaneously detects TB bacteria and genetic mutations that indicate isoniazid and/or rifampicin resistance.
Strip speciation	Strip speciation test detects a TB-specific antigen from positive liquid or solid cultures to confirm the presence of TB bacteria in culture samples.
Tools in late-stage development/evaluation
Automated detection and MDR screening	Device allows automated sample processing, DNA amplification and detection of M. tuberculosis and screening for rifampicin resistance.
Colorimetric redox Indicators	Technique detects isoniazid and rifampicin resistance in culture samples after incubation with redox dyes.
Front-loaded smear microscopy	Based on 2 or 3 specimens but aims to examine specimens on the day that patient presents to the health service (thus identifying 95% of TB cases).
Interferon gamma release assay	Blood test detects specific cellular immune responses indicating TB infection.
LED fluorescence Microscopy	Robust fluorescence microscopy (FM) systems based on light-emitting Diodes (LEDs) that could allow the advantages of FM at levels of the health system where conventional FM would be impractical.
Microscopic Observation Drug Susceptibility (MODS)	Manual liquid culture technique uses basic laboratory equipment (incl. an inverted light microscope) and microscopy skills to detect TB bacteria.
New solid culture Methods	Solid culture technique measures nitrate reduction to indicate isoniazid and rifampicin resistance. Solid culture technique simultaneously detects TB bacteria and indicates isoniazid and rifampicin resistance.
Tools in early phase of development
Breathalyser screening test
First-generation loop-mediated isothermal amplification technology platform (LAMP)
Lipoarabinomannan (LAM) detection in urine
Phage-based tests

Conclusion

The diagnosis of tuberculosis has been an ever challenging task. Despite being such a common disease, research is still going on to come up with a test that could rapidly and reliably diagnose tuberculosis in a subject. A lot of advancement has been made in this regard and newer methods providing rapid diagnosis have been formulated (Table 1). Still, mycobacterial culture remains the reference standard test. The gamut of tests available these days needs to be used in a logical way to help in reaching an accurate, rapid diagnosis without causing much financial burden on the nation. False positive diagnosis may lead to overburdened health system and excessive false negative or delayed results will not be able to curb the transmission of tuberculosis. Along with the advancement in the diagnosis, a similar pace would have to be kept by the advances in the tuberculosis treatment. Diagnosis of XDR tuberculosis is worth only if we have a treatment for it. This review article summarizes the newer and the advancements in the older diagnostic methods. Future of tuberculosis is going to change with better diagnostic modalities, treatment options with the fear of increasing drug resistance.

Authors’ Contribution

SPM: Preparation of final manuscript and submission.
SS, DK: literature search and preparation of body of manuscript.
AK: proof reading of final manuscript.
AKK: final proof reading of manuscript before submission and corresponding author.

Ethical consideration

None.

Conflict of interest

The authors declare that there are no conflict of interests.

Funding

None.

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